The objective of the proposal is to determine the molecular events involved in replication of a chemical carcinogen damaged DNA template in cultured mammalian cells treated with the proposed ultimate carcinogenic form of benzo(a)pyrene, 7,8, dihydroxy-9,l0-epoxy-7,8,9,l0-tetrahydro-benzo(a)pyrene, (B(a)P-diol-epoxide). The proposed studies make use of primary cultures of mouse epidermal cells and Vero cells (an African green monkey kidney cell line). Four specific objectives include: l) to continue to develop evidence for "gapped DNA synthesis" in epidermal cells using DNA fiber antoradiograph in order to study DNA fork progression and replicon function, 2) to determine if B(a)P-diol-epoxide DNA adducts in Vero cells act as absolute blocks to DNA replication and if not to determine the mechanism by which the whole genome is replicated, 3) to determine if B(a)P-diol-epoxide treatment of epidermal cells or Vero cells results in host cell reactivation of either UV-irradiated or B(a)P-diol-epoxide treated herpes simplex virus and whether react-ivation is mutagenic to the virus and inducible, 4) to determine at the molecular level whether there is an enhanced rate of DNA elongation which might be related to B(a)P-diol-epoxide induced reactivation of the herpes simplex virus. The rate of DNA replication will be measured by (methyl-3H) thymidine semiconservative incorporation and the rate of fork movement as well as effects on replicon initiation size will be measured by DNA fiber autoradiography. Alkaline sucrose gradient analysis and alkaline elution analysis of single-stranded newly synthesized DNA will be used to study the nature of nascent DNA in those replicons which continue to elongate DNA after B(a)P-diol-epoxide treatment. Host cell reactivation of UV or B(a)P-diol-epoxide treated herpes simplex virus will be studied by a plaque assay and the frequency of occurrence of forward mutation in the thymidine kinase gene of the virus will then be measured. A possible enhanced rate of DNA elongation in experiments will be determined by analysis of nascent DNA by both alkalin sucrose and alkaline elution analysis.